CN104762324A - A method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound - Google Patents
A method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound Download PDFInfo
- Publication number
- CN104762324A CN104762324A CN201510099327.3A CN201510099327A CN104762324A CN 104762324 A CN104762324 A CN 104762324A CN 201510099327 A CN201510099327 A CN 201510099327A CN 104762324 A CN104762324 A CN 104762324A
- Authority
- CN
- China
- Prior art keywords
- liquid
- runx2
- cell
- days
- sigma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound, particularly relates to a method of inducing a human fibroblast to transdifferentiate the human fibroblast into an osteoblast under the existence of dexamethasone by utilization of Runx2 gene expression, a low-molecular-weight compound CHIR99021 and/or a low-molecular-weight compound forskolin, and belongs to the fields of cytobiology and tissue engineering. The method includes: constructing a human skin fibroblast infected by a slow virus carrying Runx2-GFP, performing transdifferentiation of the human fibroblast under the existence of the dexamethasone by utilization of the low-molecular-weight compound CHIR99021 having a concentration of 5-10 muM and the low-molecular-weight compound forskolin having a concentration of 10 muM, changing into an osteoblast inducing liquid after 7-15 days, and inducing, and the fibroblast can be induced into the osteoblast after 5-20 days. The method can obtain osteoblasts simply and rapidly and can be used for cell therapy for bone metabolic disease, fracture, and the like. A pharmaceutical composition provided by the invention can be used for research, development and production of tissue-engineered bone, brings convenience to fundamental research and brings a prospect for clinical application.
Description
Technical field
The present invention relates to one and utilize Runx2 and the osteoblastic method of micromolecular compound induction human fibroblasts's reprogrammed, particularly one utilizes Runx2 genetic expression and micromolecular compound CHIR99021 and/or micromolecular compound forskolin, under dexamethasone existent condition, induce human fibroblasts's transdifferentiation to be osteoblastic method, belong to cytobiology and field of tissue engineering technology.
Background technology
Bone tissue engineer technology, by scleroblast and/or the biologically active factors of inducting osseous tissue regeneration can integrate and to implant in special supporting structure using as organizational project Composite Bone, wishes alternative traditional graft materials and technology.Therefore, bone tissue engineer needs a large amount of seed cell and special timbering material stick with sustenticular cell and break up.The seed cell of tradition application comprises the mescenchymal stem cell of the differentiation scleroblast in late period, scleroblast strain, marrow cell mixing or purifying.But the limited source of this type of cell, acquisition process are complicated and with the heavier injury of donor, thus widespread use is restricted.
Compared with bone marrow-derived cells, non-scleroblast as skin flbroblast abundance, be easy to obtain from patient and carry out external long-term a large amount of amplification cultivation.Utilize now cells transdifferentiate technology that skin flbroblast can have been made directly to be transformed into the functioning cell of the number of different types such as sarcoplast, neurone, liver cell, scleroblast, or be first transformed into multipotential stem cell further directed differentiation neuroblast or various dissimilar functional somatocyte again.Strikingly, these functional neuron obtained from the direct or indirect transdifferentiation of skin cells or somatocyte have no longer kept gene expression profile equimolecular and the cell characteristics of original parent cell, but obtain characteristic feature and the function of various object cell.These inducibility functioning cells not only can be used for simulating relevant disease in addition, and also successfully passing through of having is transplanted to disease animal model and is reached therapeutic purpose.
In order to realize efficient, quick, specific cells transdifferentiate, generally need to determine the assortments of genes such as special transcription factor through system, widely screening, people's cell may be more more complex than the genome credit union required for zooblast transdifferentiation under specific circumstances, some transcription factors also need corresponding signaling molecule to act synergistically could realize transdifferentiation, and the human skin cell's transdifferentiation that such as we have found NGN2 mediation just must have the synergy of two kinds of micromolecular compounds to neurocyte.The currently reported osteoblast transdifferentiationand factor can make skin flbroblast transdifferentiation be in vitro, all have the scleroblast of bone forming effect in body as BMP-2, BMP-7, LMP3 act on alone or synergistically.This several gene expression product is the protein of secretion property, and the regulation and control of its expression-secretion are complicated.Because they cause heterotopic osteogenesis by paracrine action in the non-bone tissue of surrounding, in transdifferentiated cells, long-term lasting high expression level may cause the side effect to transplantation site surrounding health tissue.
2006, ANDRE ' S J. GARCI ' A reports, by using the key transcription factor played a decisive role to scleroblast, as at the specific expressed transcription factor Runx2 of bone and its cells, not having effect for during transdifferentiation separately, the skin flbroblast transdifferentiation of rat is made to be scleroblast there being the synergy of hormone signal molecule dexamethasone.Deng Hong chief report in 2013, after having screened 10000 kinds of micromolecular compounds, found 4 kinds of small molecules, can realize the reprogrammed of skin flbroblast, wherein CHIR99021 and Forskolin has played important effect in reprogrammed process.
In addition, the cell of chemical small molecule inducing cell reprogramming transformation particular type has its unique advantage: 1. Induction Process is short, and medicine efficiently can arrive target cell; 2. controllability, accurately can control the concentration that medicine reaches needs; 3. cost is low, and chemical small molecule is once determine can scale operation.
Summary of the invention
The object of the invention is the deficiency overcoming the existence of above-mentioned prior art, propose a kind ofly to utilize required gene/small molecule combinatorial (Runx2/CHIR99021 and Forskolin), on the basis of cell expressing Runx2, combine with a certain proportion of small molecules CHIR99021 and Forskolin, induction human skin fibroblast is to osteoblast differentiation.
Another object of the present invention is to provide a kind of pharmaceutical composition for preventing or treat the disease relevant to osteoblast differentiation, early stage is in the combination of CHIR99021 and the 10 μm Forskolin of high density 9 μm, the expression of the Runx2 certain time keeping external source to import, under the synergy of dexamethasone, to scleroblast direction induction.
The present invention completes the object of the invention by the following technical solutions: one utilizes Runx 2 and micromolecular compound induction human fibroblasts reprogrammed to be osteoblastic method, it is characterized in that the expression and micromolecular compound CHIR99021 and forskolin that utilize Runx2 gene, induction human fibroblasts transdifferentiation is scleroblast.
First build the slow virus of carrying Runx2-GFP and contaminate human skin fibroblast, normal human skin inoblast is made to express Runx2, placing Nostoc commune Vanch liquid cultivates after 3 ~ 7 days, dexamethasone two kinds of materials of forskolin+5 ~ 15nM of micromolecular compound 6 μMs ~ 12 μMs are added again at this Nostoc commune Vanch liquid, composition hormone signal molecule induced liquid, the cell cultures of 7 ~ 15 days is carried out again in this hormone signal molecule induced liquid, osteoblast differentiation liquid is utilized to carry out inducing (37 ° of C, 5%CO subsequently
2), can induce after 5 ~ 7 days as scleroblast;
Described Nostoc commune Vanch liquid refers to: 10% foetal calf serum+100U/ml penicillin+100 μ g/ml Streptomycin sulphate+high sugared DMDM substratum, at 37 ° of C, 5%CO
2culturing cell under environment;
Described osteoblast differentiation liquid formula is: 1
0% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco)+10mM β-Phosphoric acid glycerol esters+100nM dexamethasone+0.2mM ascorbate-2-phosphate; At 37 ° of C, 5%CO
2culturing cell under environment.
Described hormone signal molecule induced liquid can also be add the micromolecular compound CHIR99021 material of 5 ~ 10 μMs again at Nostoc commune Vanch liquid, then add by CHIR99021(5 ~ 10 μM at Nostoc commune Vanch liquid)+forskolin(6 μM ~ 12 μMs) namely mixed solution that+dexamethasone (5 ~ 15nM) three mixed solution forms is hormone signal molecule induced liquid.
In composition hormone signal molecule induced liquid, optimum quantity is: 9uM micromolecular compound CHIR99021+10uM micromolecular compound forskolin+10nM dexamethasone.
Described utilizes Runx2 and the osteoblastic method of micromolecular compound induction human fibroblasts's reprogrammed, and step is
A. gathered scleroblast is obtained by the direct transdifferentiation of the expression of transcription factor Runx2 by the general population's skin cells, this skin cells does not need traditional inductive pluripotent stem cells (iPS) four factor (c-Myc, Klf4, Oct4, Sox2), thus without stem cell stage, when avoiding transplanting, knurl risk is become;
B. the dexamethasone in utilized hormone signal molecule induced liquid and micromolecular compound CHIR99021 and/or the combination of micromolecular compound forskolin under finite concentration, the human skin fibroblast that the above-mentioned Runx2 that arranges in pairs or groups expresses, and maintain the certain expression time window 11 days ~ 15 days of Runx2, and make (transdifferentiation starts rear after 6 ~ 11 days) alkaline phosphatase (ALP) expression in cell in early days, this one-tenth bone seeding cell can be obtained;
Described alkaline phosphatase (ALP) refers to: one of osteoblastic phenotypic marker, the albumen of early expression in the process of Osteoblast Differentiation.
Concrete steps are:
One, plasmid construction and cell cultures
1.1 cell cultures
First gathering the general population skin flbroblast, human skin fibroblast to be incubated in the Nostoc commune Vanch liquid (Gibco) containing 10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM composition and in adherent 6 orifice plates in being coated with gelatin; Inoculating cell density 5 ~ 6x10
3/ cm
2be incubated at 37 DEG C, in the incubator of 5% CO2;
1.2 plasmid construction
The cDNA of total length Runx2 (deriving from the mankind) is inserted in the pVSVG carrier containing GFP, also has the zero load of the pVSVG only containing GFP in addition;
Two, virus infection cell
Slow virus packaging and infection: goal gene (Runx2) plasmid and viral packaging plasmid are by Lipofectamine 2000 cotransfection HEK293T cell, obtain the virus of high titre, respectively to the slow virus of this cell infection Runx2-GFP-PVSVG slow virus and GFP-PVSVG zero load, efficiency of infection can reach 61% and 93.4% respectively;
Three, small molecules induction
Skin flbroblast after virus infection is cultivated 3 ~ 7 days, use Nostoc commune Vanch liquid to cultivate, formula is: 10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco);
Be replaced by the hormone signal molecule inductor formed after Nostoc commune Vanch liquid is added with micromolecular compound afterwards and cultivate 7 ~ 15 days again, hormone signal molecule inductor comprises the CHIR99021(concentration range of 10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco)+9 μMs: 5 ~ 10 μMs)+10 μMs of Forskolin (concentration range: 6 μMs ~ 12 μMs)+10nM dexamethasone;
Four, Osteoblast Differentiation nutrient solution
Be replaced by osteoblast differentiation nutrient solution subsequently again to cultivate, osteoblast differentiation liquid comprises
10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco)+10mM β Phosphoric acid glycerol esters+100nM dexamethasone+0.2mM ascorbate-2-phosphate; after this; an osteoblast differentiation liquid is changed, successive induction more than 5 days every 2 ~ 3 days; Thereafter cell cultures, uses osteoblast differentiation liquid to maintain always;
Five, Alizarin red staining qualification
1. old induced liquid is absorbed, the cell after induction is exposed, utilize PBS to clean cell 3 times;
2. the stationary liquid fixed cell 30min containing 4% paraformaldehyde is got;
3. remove stationary liquid, utilize the sodium alizarinsulfonate dye liquor of 2%, 37 DEG C, hatch 30min, can be placed in incubator and carry out;
4. discard Alizarin red staining liquid, utilize ultrapure water to clean more than 3 times, to avoid occurring false positive
5. to take pictures under inverted microscope experimental group and control group.
The scleroblast that the present invention gathers is obtained by the direct transdifferentiation of the expression of transcription factor Runx2 by the general population's skin cells, this skin cells does not need traditional inductive pluripotent stem cells (iPS) four factor (c-Myc, Klf4, Oct4, Sox2), thus without stem cell stage, when avoiding transplanting, knurl risk is become.
Human skin fibroblast can turn and point is divided into scleroblast by the inventive method simply, fast, then obtain and obtain inducibility scleroblast specifically, it can promoting bone growing and contribute to treat various osteonosus, such as osteoporosis, the bone metabolism diseases such as fracture.
Accompanying drawing explanation
Fig. 1 the present inventor skin flbroblast transdifferentiation is osteoblastic method schematic diagram.
Fig. 2 is that Runx2 of the present invention infects human skin fibroblast efficiency.
Fig. 3 is the present inventor's skin flbroblast transdifferentiation is osteoblastic qualification.In figure 3: a, the fibroblastic cultivation form of normal human skin; B, transdifferentiation is osteoblastic cellular form (green fluorescence represents); C, in transdifferentiation process, Cellular alkaline phosphatase dyes; D, transdifferentiation is the Mineral nodules (agglomerate is red) formed after scleroblast; E, ALP(alkaline phosphatase) and the expression identification of Runx2 gene.
Fig. 4 is the subcutaneous transplantation that the cell of transdifferentiation carries out mouse, forms area of new bone.A in the diagram, after cell mouse subcutaneous transplantation, the area of new bone (in circle) of formation; B, a seen under white light scheme the section qualification of graft; C, a that UV-light is seen scheme the section qualification of graft.
Embodiment
The present invention utilizes expression and micromolecular compound CHIR99021 and forskolin of Runx2 gene, induces human fibroblasts's transdifferentiation to be osteoblastic process to be:
(1) the general population's skin flbroblast is gathered, cultivator skin flbroblast;
(2) Runx2 lentiviral vectors is built;
(3) slow virus infection human skin fibroblast, is specified to fibrocyte and starts to express Runx2;
(4) infect after 3-7 days, cell density 2 ~ 3x10
4when/cm2, with the CHIR99021+10 of 9 μm μm Forskolin and add 10nM dexamethasone hormone signal molecule liquid process cell 7 ~ 15 days;
(5) add scleroblast induced liquid after to induce, continue can obtain scleroblast in 10 ~ 20 days; Cell cultures can be maintained with this induced liquid;
(6) obtain scleroblast, and identify.
Present method uses small molecules (forskolin 6 μm ~ 12 μm+dexamethasone, 5 ~ 15nM) that the human skin fibroblast transdifferentiation expressing Runx2 also can be made to be scleroblast, although the height that transformation efficiency (forming skeletonization mark: the ratio of Mineral nodules is not high) exists not as three's (forskolin+CHIR99021+dexamethasone) compound, but first introduce the use of small molecules forskolin, be beneficial to human skin fibroblast and break up to scleroblast direction.
embodiment 1
One, plasmid construction and cell cultures
1.1 human skin fibroblast is cultivated: first gather the general population skin flbroblast, human skin fibroblast to be incubated in the Nostoc commune Vanch liquid (Gibco) containing 10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM composition and in adherent 6 orifice plates in being coated with gelatin; Inoculating cell density 5 ~ 6x10
3/ cm
2be incubated at 37 DEG C, in the incubator of 5% CO2.
1.2 build Runx2 lentiviral vectors: be inserted into by the cDNA of total length Runx2 (deriving from the mankind) in the pVSVG carrier containing GFP, also build the zero load of the pVSVG only containing GFP in addition.
Two, virus infection cell
Slow virus packaging and infection: goal gene Runx2 plasmid and viral packaging plasmid are by Lipofectamine 2000 cotransfection HEK293T cell, obtain the virus of high titre, infect Runx2-GFP-PVSVG slow virus and the unloaded slow virus of GFP-PVSVG respectively to common human skin fibroblast, efficiency can reach 61% and 93.4% respectively.
Three, small molecules induction
By the human skin fibroblast (now cell expresses Runx2 or GFP respectively) after virus infection, cultivate 3 ~ 7 days, use Nostoc commune Vanch liquid, this nutrient solution comprises 10% foetal calf serum (Hyclone), 100U/ml penicillin (Sigma), 100 μ g/ml Streptomycin sulphates (Sigma), high sugared DMDM (Gibco).
Be replaced by hormone signal molecule induced liquid afterwards and cultivate 7 ~ 15 days again, hormone signal molecule induced liquid comprises 10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco)+10 μMs Forskolin μ μ+10nM dexamethasone;
Four, Osteoblast Differentiation nutrient solution
After 7 ~ 15 days, then be replaced by osteoblast differentiation liquid and cultivate, this osteoblast differentiation nutrient solution comprises
10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco)+10mM β-Phosphoric acid glycerol esters+100nM dexamethasone+0.2mM ascorbate-2-phosphate; after this; an osteoblast differentiation nutrient solution is changed, successive induction more than 5 days every 2 ~ 3 days; Thereafter cell cultures, uses Osteoblast Differentiation liquid to maintain always.
Five, Alizarin red staining qualification
1. old induced liquid is absorbed, the cell after induction is exposed, utilize PBS to clean cell 3 times;
2. the stationary liquid fixed cell 30min containing 4% paraformaldehyde is got;
3. remove stationary liquid, utilize the sodium alizarinsulfonate dye liquor of 2%, 37 DEG C, hatch 30min, can be placed in incubator and carry out;
4. discard Alizarin red staining liquid, utilize ultrapure water to clean more than 3 times, to avoid occurring false positive;
5. to take pictures under inverted microscope experimental group and control group.
embodiment 2~
embodiment8: identical with embodiment 1 with transdifferentiation method to cultivation in embodiment 2 to embodiment 8, no longer emphasis is stated, but its formula is different with the operating time, shown in table specific as follows.
Claims (6)
1. one kind utilizes Runx 2 and micromolecular compound induction human fibroblasts reprogrammed to be osteoblastic method, it is characterized in that the expression and micromolecular compound CHIR99021 and forskolin that utilize Runx2 gene, induction human fibroblasts transdifferentiation is scleroblast.
2. method according to claim 1, it is characterized in that first building the slow virus of carrying Runx2-GFP and contaminate human skin fibroblast, normal human skin inoblast is made to express Runx2, placing Nostoc commune Vanch liquid cultivates after 3 ~ 7 days, dexamethasone two kinds of materials of forskolin+5 ~ 15nM of micromolecular compound 6 μMs ~ 12 μMs are added again at this Nostoc commune Vanch liquid, composition hormone signal molecule induced liquid, the cell cultures of 7 ~ 15 days is carried out again in this hormone signal molecule induced liquid, osteoblast differentiation liquid is utilized to carry out inducing (37 ° of C, 5%CO subsequently
2), can induce after 5 ~ 7 days as scleroblast;
Described Nostoc commune Vanch liquid refers to: 10% foetal calf serum+100U/ml penicillin+100 μ g/ml Streptomycin sulphate+high sugared DMDM substratum, at 37 ° of C, 5%CO
2culturing cell under environment;
Described osteoblast differentiation liquid formula is: 1
0% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco)+10mM β-Phosphoric acid glycerol esters+100nM dexamethasone+0.2mM ascorbate-2-phosphate; At 37 ° of C, 5%CO
2culturing cell under environment.
3. method according to claim 2, it is characterized in that: described hormone signal molecule induced liquid can also be add the micromolecular compound CHIR99021 material of 5 ~ 10 μMs again at Nostoc commune Vanch liquid, then add by CHIR99021(5 ~ 10 μM at Nostoc commune Vanch liquid)+forskolin(6 μM ~ 12 μMs) namely mixed solution that+dexamethasone (5 ~ 15nM) three mixed solution forms is hormone signal molecule induced liquid.
4. Runx2 and the micromolecular compound of utilizing according to claim 1 and 2 induces the osteoblastic method of human fibroblasts's reprogrammed, it is characterized in that: in composition hormone signal molecule induced liquid, optimum quantity is: 9uM micromolecular compound CHIR99021+10uM micromolecular compound forskolin+10nM dexamethasone.
5. Runx2 and the micromolecular compound of utilizing according to claim 1 and 2 induces the osteoblastic method of human fibroblasts's reprogrammed, it is characterized in that:
A. gathered scleroblast is obtained by the direct transdifferentiation of the expression of transcription factor Runx2 by the general population's skin cells, this skin cells does not need traditional inductive pluripotent stem cells (iPS) four factor (c-Myc, Klf4, Oct4, Sox2), thus without stem cell stage, when avoiding transplanting, knurl risk is become;
B. the dexamethasone in utilized hormone signal molecule induced liquid and micromolecular compound CHIR99021 and/or the combination of micromolecular compound forskolin under finite concentration, the human skin fibroblast that the above-mentioned Runx2 that arranges in pairs or groups expresses, and maintain the certain expression time window 11 days ~ 15 days of Runx2, and make (transdifferentiation starts rear after 6 ~ 11 days) alkaline phosphatase (ALP) expression in cell in early days, this one-tenth bone seeding cell can be obtained;
Described alkaline phosphatase (ALP) refers to: one of osteoblastic phenotypic marker, the albumen of early expression in the process of Osteoblast Differentiation.
6. Runx2 and the micromolecular compound of utilizing according to claim 1 and 2 induces the osteoblastic method of human fibroblasts's reprogrammed, and its characteristic sum concrete steps are
(1), plasmid construction and cell cultures
1.1 cell cultures
First gathering the general population skin flbroblast, human skin fibroblast to be incubated in the Nostoc commune Vanch liquid (Gibco) containing 10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM composition and in adherent 6 orifice plates in being coated with gelatin; Inoculating cell density 5 ~ 6x10
3/ cm
2be incubated at 37 DEG C, in the incubator of 5% CO2;
1.2 plasmid construction
The cDNA of total length Runx2 (deriving from the mankind) is inserted in the pVSVG carrier containing GFP, also has the zero load of the pVSVG only containing GFP in addition;
(2), virus infection cell
Slow virus packaging and infection: goal gene (Runx2) plasmid and viral packaging plasmid are by Lipofectamine 2000 cotransfection HEK293T cell, obtain the virus of high titre, respectively to the slow virus of this cell infection Runx2-GFP-PVSVG slow virus and GFP-PVSVG zero load, efficiency of infection can reach 61% and 93.4% respectively;
(3), small molecules induction
Skin flbroblast after virus infection is cultivated 3 ~ 7 days, use Nostoc commune Vanch liquid to cultivate, formula is: 10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco);
Be replaced by the hormone signal molecule inductor formed after Nostoc commune Vanch liquid is added with micromolecular compound afterwards and cultivate 7 ~ 15 days again, hormone signal molecule inductor comprises the CHIR99021(concentration range of 10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco)+9 μMs: 5 ~ 10 μMs)+10 μMs of Forskolin (concentration range: 6 μMs ~ 12 μMs)+10nM dexamethasone;
(4), Osteoblast Differentiation nutrient solution
Be replaced by osteoblast differentiation nutrient solution subsequently again to cultivate, osteoblast differentiation liquid comprises
10% foetal calf serum (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomycin sulphate (Sigma)+high sugared DMDM substratum (Gibco)+10mM β Phosphoric acid glycerol esters+100nM dexamethasone+0.2mM ascorbate-2-phosphate; after this; an osteoblast differentiation liquid is changed, successive induction more than 5 days every 2 ~ 3 days; Thereafter cell cultures, uses osteoblast differentiation liquid to maintain always;
Five, Alizarin red staining qualification
1. old induced liquid is absorbed, the cell after induction is exposed, utilize PBS to clean cell 3 times;
2. the stationary liquid fixed cell 30min containing 4% paraformaldehyde is got;
3. remove stationary liquid, utilize the sodium alizarinsulfonate dye liquor of 2%, 37 DEG C, hatch 30min, can be placed in incubator and carry out;
4. discard Alizarin red staining liquid, utilize ultrapure water to clean more than 3 times, to avoid occurring false positive
5. to take pictures under inverted microscope experimental group and control group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510099327.3A CN104762324B (en) | 2015-03-06 | 2015-03-06 | Method using Runx2 and micromolecular compound induction human fibroblasts reprogramming for Gegenbaur's cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510099327.3A CN104762324B (en) | 2015-03-06 | 2015-03-06 | Method using Runx2 and micromolecular compound induction human fibroblasts reprogramming for Gegenbaur's cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104762324A true CN104762324A (en) | 2015-07-08 |
| CN104762324B CN104762324B (en) | 2017-12-15 |
Family
ID=53644415
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510099327.3A Active CN104762324B (en) | 2015-03-06 | 2015-03-06 | Method using Runx2 and micromolecular compound induction human fibroblasts reprogramming for Gegenbaur's cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104762324B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062963A (en) * | 2015-03-23 | 2015-11-18 | 昆明学院 | Culture system for promoting transdifferentiation of human skin fibroblasts to osteoblasts |
| CN107217033A (en) * | 2017-07-14 | 2017-09-29 | 陈强 | A kind of utilization fish oocyte extract induced fibroblast reprogramming is the kit and method of osteoblast-like cells |
| CN108060121A (en) * | 2016-11-07 | 2018-05-22 | 昆明学院 | Micromolecular compound combines and is combined using the micromolecular compound method that the cell of differentiation is induced to prepare osteoblast |
| CN108300686A (en) * | 2017-01-13 | 2018-07-20 | 云南美颜美龄生物科技有限公司 | For preventing, delaying or micromolecular compound/combination of reverse both, tissue, organ, body aging, product and application thereof |
| CN108611316A (en) * | 2018-05-04 | 2018-10-02 | 江南大学附属医院 | A method of induction human fibroblasts transdifferentiation is human germ-line |
| CN110093310A (en) * | 2018-01-29 | 2019-08-06 | 中国科学院动物研究所 | A kind of method and its application converting fibroblast to immortalized cells |
| CN110093309A (en) * | 2018-01-29 | 2019-08-06 | 中国科学院动物研究所 | A kind of induced fibroblast transdifferentiation is the method for fat cell |
| CN113846054A (en) * | 2021-09-23 | 2021-12-28 | 四川大学 | Matrix material for directly reprogramming fibroblasts into osteoblasts, preparation method and reprogramming method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010124290A2 (en) * | 2009-04-24 | 2010-10-28 | Whitehead Institute For Biomedical Research | Compositions and methods for deriving or culturing pluripotent cells |
| CN104278008A (en) * | 2013-07-12 | 2015-01-14 | 北京大学科技开发部 | Method, kit and applications of preparing pluripotent stem cells through small-molecule compound treatment |
-
2015
- 2015-03-06 CN CN201510099327.3A patent/CN104762324B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010124290A2 (en) * | 2009-04-24 | 2010-10-28 | Whitehead Institute For Biomedical Research | Compositions and methods for deriving or culturing pluripotent cells |
| CN104278008A (en) * | 2013-07-12 | 2015-01-14 | 北京大学科技开发部 | Method, kit and applications of preparing pluripotent stem cells through small-molecule compound treatment |
Non-Patent Citations (2)
| Title |
|---|
| JENNIFER E. PHILLIPS等: "Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation", 《JOURNAL OF CELL SCIENCE》 * |
| 王艳艳等: "成纤维细胞直接重编程为心肌细胞的研究进展", 《中国细胞生物学学报》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062963A (en) * | 2015-03-23 | 2015-11-18 | 昆明学院 | Culture system for promoting transdifferentiation of human skin fibroblasts to osteoblasts |
| CN105062963B (en) * | 2015-03-23 | 2018-05-25 | 云南济慈再生医学研究院有限公司 | A kind of promotion cultural method of the human skin fibroblasts to osteoblast transdifferentiationand |
| CN108060121A (en) * | 2016-11-07 | 2018-05-22 | 昆明学院 | Micromolecular compound combines and is combined using the micromolecular compound method that the cell of differentiation is induced to prepare osteoblast |
| CN108300686A (en) * | 2017-01-13 | 2018-07-20 | 云南美颜美龄生物科技有限公司 | For preventing, delaying or micromolecular compound/combination of reverse both, tissue, organ, body aging, product and application thereof |
| CN108300686B (en) * | 2017-01-13 | 2022-08-26 | 云南美颜美龄生物科技有限公司 | Small molecule compound/combination for preventing, delaying or reversing cell, tissue, organ and organism aging, product and application thereof |
| CN107217033A (en) * | 2017-07-14 | 2017-09-29 | 陈强 | A kind of utilization fish oocyte extract induced fibroblast reprogramming is the kit and method of osteoblast-like cells |
| CN110093310A (en) * | 2018-01-29 | 2019-08-06 | 中国科学院动物研究所 | A kind of method and its application converting fibroblast to immortalized cells |
| CN110093309A (en) * | 2018-01-29 | 2019-08-06 | 中国科学院动物研究所 | A kind of induced fibroblast transdifferentiation is the method for fat cell |
| CN110093310B (en) * | 2018-01-29 | 2021-07-02 | 中国科学院动物研究所 | Method for converting fibroblasts into immortalized cells and application thereof |
| CN108611316A (en) * | 2018-05-04 | 2018-10-02 | 江南大学附属医院 | A method of induction human fibroblasts transdifferentiation is human germ-line |
| CN113846054A (en) * | 2021-09-23 | 2021-12-28 | 四川大学 | Matrix material for directly reprogramming fibroblasts into osteoblasts, preparation method and reprogramming method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104762324B (en) | 2017-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104762324A (en) | A method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound | |
| CN104726406B (en) | It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell | |
| CN106102754A (en) | Cancer treatment medical composition and using said composition as the cancer treatment pharmaceutical preparation of effective ingredient | |
| CN104232574A (en) | Method for in-vitro directional differentiation inducing of mesenchymal stem cell towards melanocyte | |
| CN104587528A (en) | Acellular matrix of human heart valve tissue and preparation and application of acellular matrix | |
| CN103814124A (en) | In vitro cardiovascular model | |
| CN104774808A (en) | Method for inducible differentiation of umbilical cord mesenchymal stem cells into gamma-aminobutyric acid-ergic neuron | |
| CN105039247A (en) | Preparation used for inducing stem cells to differentiate towards chondrogenesis and preparation method and application of preparation | |
| CN102161980B (en) | Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast | |
| CN101372682B (en) | Construction method of Epinephelus fuscoguttatus fin cell line | |
| CN104250655A (en) | BMP(bone morphogenetic protein)-2/VEGF(vascular endothelial growth factor)165 double gene modified bone marrow mesenchymal stem cells and preparation method thereof | |
| CN101497874B (en) | Method for promoting somatic cell proliferation | |
| CN1884494A (en) | Method for inducing human embryo stem cell differentiation to liver cell and the special-purpose medium | |
| CN104789531B (en) | A kind of method that umbilical cord mesenchymal stem cells are induced differentiation into dopaminergic neuron | |
| CN101712947A (en) | Preparation method and application of mesenchymal stem cells deriving from embryonic stem cells | |
| US11186828B2 (en) | Method of making human cells expressing OCT4, SOX2, and Nanog using an Ecklonia cava extract | |
| CN100540659C (en) | A kind of method and special culture media thereof of cultivating mouse embryo stem cell | |
| CN107287158A (en) | The method of effective acquisition mescenchymal stem cell from mouse dense bone | |
| Carvalho et al. | Could the coculture of skeletal myoblasts and mesenchymal stem cells be a solution for postinfarction myocardial scar? | |
| CN106282101A (en) | A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application | |
| CN106434543A (en) | Culture medium and cell cultural method | |
| CN102796698A (en) | Method for inducing differentiation of placenta-derived mesenchymal stem cells into islet-like cells | |
| CN105062963B (en) | A kind of promotion cultural method of the human skin fibroblasts to osteoblast transdifferentiationand | |
| CN107574147A (en) | A kind of mescenchymal stem cell Proliferation, Differentiation nutrient solution using atractylenolide as trophic factors | |
| CN104450622B (en) | Recombinant cell line, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20180320 Address after: 650217 Yunnan Province, Kunming Economic Development Zone Export Processing Zone Shuntong road Ginza international B3 Building 29 room 2907 Patentee after: Yunnan Keats Institute of Regenerative Medicine Co., Ltd. Address before: 650214 medical building of Kunming college, No. 2, Puxin Road, Kunming economic and Technological Development Zone, Yunnan Patentee before: Kunming University |
|
| TR01 | Transfer of patent right |